Abstract

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

Inducible Gpx4 disruption causes ARF and death in mice. (a) A scheme showing the most important steps of glutathione (GSH) biosynthesis. αToc, α-tocopherol; BSO, L-buthionine sulphoximine; GSSG, oxidized glutathione; γGCS, γ-glutamylcysteine-synthase. (b) Inhibitors against enzymes of arachidonic acid metabolism prevent Gpx4-deletion-induced cell death in a dose-dependent manner. Gpx4 was disrupted in Pfa1 cells by the addition of 1 μM TAM in the presence of increasing concentrations of inhibitors. Cell viability was assessed by using AquaBluer 72 h after knockout induction. Data shown represent the mean ± s.d. of n = 4 of a 96-well plate from a representative experiment wells performed independently four times. (c) Mouse survival after TAM feeding. All induced Gpx4−/− (KO) mice died after approximately 2 weeks of TAM feeding regardless of Alox15 expression. None of the control mice (CreERT2;Gpx4+/fl/Alox15+/+(WT/Alox15+/+), CreERT2;Gpx4+/fl/Alox15−/−(WT/Alox15−/−)) died in the period investigated. Data are percentage of live animals; mean survival of Gpx4-null mice is 13.5 days after the onset of TAM feeding (n = 8 animals for KO/Alox15−/− and WT/Alox15−/− and n = 19 animals for KO/Alox15+/+ and WT/Alox15+/+). Gehan–Breslow–Wilcoxon test: P <0.0001). (d) Overall kidney phenotype of TAM-treated CreERT2;Gpx4fl/fl animals (KO) at time of euthanization. Left, control kidney (TAM-treated CreERT2;Gpx4+/fl, WT); right, enlarged and pale Gpx4−/− kidney. (e) Western blot of whole kidney tissue extracts showing that Gpx4 was efficiently depleted on TAM feeding in CreERT2;Gpx4fl/fl (fl/fl,Cre), but not in control CreERT2;Gpx4+/fl (+/fl,Cre) mice. (f) TAM-inducible CreERT2;Gpx4fl/fl mice present massive albuminuria and unselective proteinuria compared with control mice. Each lane represents one knockout or control animal (A, murine albumin). (g) Immunohistochemical expression analysis of Gpx4 in kidney tissue revealed that Gpx4 was efficiently depleted on TAM treatment of CreERT2;Gpx4fl/fl mice, which is in line with the immunoblot data. Note the high expression of Gpx4 in tubule cells of kidney cortex, whereas glomeruli show only faint Gpx4 expression (bars top row 100 μm and bottom row 50 μm). (h) Histological analysis of kidneys of TAM-treated CreERT2;Gpx4fl/fl animals showed widespread tubular cell death, interstitial edema and proteinaceous casts in distal tubules (bars top row 100 μm and bottom row 50 μm). (i) The number of TUNEL+ cells and mitotic cells (phosho-histone H3 staining, PH-3) is increased in kidneys of symptomatic Gpx4−/− mice (bars 100 μm). Uncropped images of blots are shown in .

Ferroptosis in human cells and in murine disease models can be targeted by Liproxstatin-1. (a) HRPTEpiCs are susceptible to Gpx4-inhibition-induced ferroptosis, which can be prevented by Liproxstatin-1 (100 nM). Dose-dependent killing of HRPTEpiCs by active (1S,3R)-RSL3 in contrast to inactive (1R,3R)-RSL3. Viability was assessed 24 h after treatment using AquaBluer. Data shown represent the mean ± s.d. of n = 3 wells of a 96-well plate from a representative experiment performed independently three times. (b) Liproxstatin-1 prevents RSL3 (0.2 μM)-induced lipid oxidation in HRPTEpiCs. Lipid peroxidation was assessed 24 h after knockout induction using the redox-sensitive dye BODIPY 581/591 C11. A representative experiment is shown performed independently four times. (c) Liproxstatin-1 retards ARF and death of mice induced by Gpx4 deletion; median survival was calculated to be 11 days for vehicle-treated (n = 12) and 14 days for Liproxstatin-1-treated mice (n = 13), Gehan–Breslow–Wilcoxon test: P <0.0001. Representative experiment shown was performed two times. Mice were injected daily with Liproxstatin-1 (10 mg kg−1, i.p.) during the course of the experiment. (d) Quantification of TUNEL cells in kidneys of vehicle- and Liproxstatin-1-treated animals at 9 +days after TAM administration. Data shown represent the mean ± s.d. of n = 4 comparable anatomical sections from a representative experiment performed two times (scale bars 50 μm). (e) The extent of tissue injury on transient ischaemia/reperfusion in liver of C57BL/6J mice can be ameliorated by the ferroptosis inhibitor Liproxstatin-1 as measured by AST/ALT (n = 17) for vehicle and for Liproxstatin-1 each) and by determining the necrotic area (n = 5). Data represent the mean ± s.e.m.; ∗P = 0.05 or ∗∗∗P = 0.001 (one-way ANOVA) followed by Dunnett’s post-test.